J. Nannoplankton Res., 41(2), 2023, pp. 98–104 https://doi.org/10.58998/jnr3439© 2023 The Authors ISSN 1210-8049 Printed by The Sheridan Press, USA

98

Molecular phylogenetic affinity of Hayaster cf. perplexus
based on 18S rDNA sequences
Kyoko Hagino*

Kochi Core Institute, Kochi University, Monobe B200, Nankoku, Kochi, 783-8522 Japan: hagino@kochi-u.ac.jp

Jeremy R. Young

Earth Sciences, University College London, Gower St., London WC1E 6BT, UK

Yuki Morono

Kochi Institute for Core Sample Research, Extra-cutting-edge Science and Technology Avant-garde Research (X-star),
Japan Agency for Earth-Marine Science and Technology (JAMSTEC), Monobe B200, Nankoku, Kochi, 783-8522 Japan*Corresponding author
Manuscript received 23 June 2023; revised manuscript accepted 21 August 2023
Available online 25 October 2023
DOI: 10.58998/jnr3439
This is an open access article licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license(https://creativecommons.org/licenses/by/4.0/).

Abstract A partial 18S rDNA sequence of Hayaster cf. perplexus (1674 bp) was obtained from an isolated cellfrom Tosa Bay, off the coast of Japan (NW Pacific Ocean), using the single cell PCR technique. In molecular phylogenetic analyses, the sequence of H. cf. perplexus fell in the Coccolithales clade and clustered with the sequencesof the Coccolithaceae, Calcidiscaceae, an uncultured clone, and holococcolith-bearing taxa with very high posterior
probability for Bayesian Inference analyses and very high bootstrap support for PhyML analyses. However, phylogenetic relationships among the sequences of H. cf. perplexus, Coccolithaceae, and Calcidiscaceae are uncertain due
to low posterior probability and bootstrap values. These results suggest that the separation of the families Coccolithaceae and Calcidiscaceae may not be justified.

Keywords Coccolithophore, Calcidiscaceae, Coccolithaceae, Hayaster, molecular phylogeny
1. Introduction
Coccolithophores are unicellular marine haptophytes characterised by calcified scales called coccoliths. The classification of coccolithophores is based on the morphology of the coccoliths and the crystallographic orientationof the calcite elements that form them (e.g. Young et al.,2003). Molecular phylogenetic studies of coccolithophores
mainly based on 18S rDNA sequences from culture strains
started in the late 1990s, and, whilst providing much new
information on phylogenetic relationships, they largely
supported the classical morphological and crystallographic
classification (e.g. Medlin et al., 2008; Edvardsen et al.,2011). In addition, at the species level, phylogenetic studies based on other genetic regions or on genomic analysesof culture strains revealed that fine morphological variationof coccoliths within several morpho-species are often the
result of pseudo-cryptic speciation (e.g. Sáez et al., 2003;
Geisen et al., 2004; Bendif et al., 2023). Molecular data are
therefore a powerful tool for understanding the taxonomy,
phylogeny, and diversity of coccolithophores, although thefeasibility of analyses fundamentally depends on availability of culture strains. Less than one quarter of the ~300
described species have been cultured and sequenced, so themolecular phylogenetic tree of the coccolithophores lacks
most uncultured species.
Molecular data from uncultured phytoplankton can beobtained using the single cell PCR technique (Takano &Horiguchi, 2004), although the sequence regions that canbe determined from single cells are limited. Takano et al.
(2006) were the first to apply the single cell PCR techniqueto coccolithophores. They determined nearly complete 18S
rDNA sequences of Braarudosphaera bigelowii from twocells, and found differences in the size of the pentaliths
and in 18S rDNA sequences between the cells. Subsequentstudies based on the single cell PCR technique revealed
relationships between 18S rDNA genotypes and size morphotypes of B. bigelowii (Hagino et al., 2009), the 16S
rDNA genotype of the endosymbiont (nitrogen-fixing cyanobacterium UCYN-A) in the B. bigelowii 18S genotype
III (Hagino et al., 2013), and the phylogenetic positions

99

Hagino, Young, Morono

of Gladiolithus sp. (Young et al., 2014) and of Tergestiella adriatica (Hagino et al., 2015). Thus, the single-cell
PCR technique is useful for the study of selected genetic
regions of uncultured coccolithophores.
While studying coccolithophores in seawater from
Tosa Bay off the coast of Japan (NW Pacific Ocean), we
encountered an unfamiliar coccoid cell using an inverted
light microscope. Since the condition of the cell did notappear good enough for culture experiments, we applied
the single-cell PCR technique to it. Here we report the results of morphological and molecular phylogenetic studiesof the cell.
2. Materials and Methods
A 10 L surface seawater sample was collected for microscopic and molecular studies of coccolithophores from
Tosa Bay (33°15’N, 133°28’E) using a bucket from theboat Neptune of the Usa Marine Biological Institute, Kochi University (Japan) on 16 October 2014. The sample
was pre-filtered through a 50 µm mesh size plankton net(Sefar Inc. NY50-HD), and then concentrated using a 1µm mesh size plankton net (Sefar Inc. NY1-HD). One of
the unidentified coccoid cells, labelled as Tosa-3, was isolated using a capillary tube under an inverted light microscope (LM) (Olympus CKX41). The isolate was placed ona glass slide with a drop of sterile seawater, covered witha glass coverslip, and then photographed under plane- andcross-polarised light using a Nikon DS-Fi2 digital camera
equipped on a Nikon E600POL microscope. After photography, the coverslip was removed and the coccosphere
was re-isolated using a capillary tube under the inverted
LM, then subjected to single cell PCR amplification as
outlined in Takano & Horiguchi (2004).
The first round of PCR was performed using external
primers (SR1 and SR12) to amplify almost the entire 18S
rDNA sequence. The PCR condition for the first round
was one initial denaturation at 94°C for 60 s, followed by
35 cycles of denaturation at 94°C, annealing at 50°C for
30 s, and an extension cycle at 72°C for 60 s. The temperature profile was completed by a final extension cycle
at 72°C for 4 min. In the second round of PCR, internal
short sequences of 18S rDNA region were amplified using the 0.5 µL of the first round PCR product as a DNA
template with three pairs of internal primers: SR1c–SR5,
SR4–SR9, and SR8tak–SR12b. The sequences of primers used in this study were listed in Takano & Horigu-chi (2004). The PCR condition for the second round was
one initial denaturation step at 94°C for 30 s, followed by
35 cycles of denaturation at 94°C for 30 s, annealing at
55°C for 30 s, and an extension cycle at 72°C for 30 s.
The temperature profile was completed by a final extension cycle at 72°C for 4 min. The products of the second
round PCR amplification were sequenced directly using
the BigDye Terminator Cycle Sequencing Kit ver. 3.1(ThermoFisher Scientific) with the DNA auto sequencer
ABI PRISM 3130 Genetic Analyzer (Applied Biosystem)
in Kochi Institute for Core Sample Research, JAMSTEC.
The quality of each sequence result was checked and ambiguous results (~10-30-bp at the 5’ and 3’ ends of eachsequence) were removed using the software 4Peaks (Mekentosj). The accuracy of the sequence results was confirmed based on the sequences obtained by forward andreverse primers for the same genetic region being identicalon SeaView 5.04 (Manolo Gouy) (Gouy et al., 2009). A
partial 18S rDNA sequence of the isolate Tosa-3 (1674bp)(Accession number LC771592) was reconstructed by connecting the confirmed sequences on SeaView 5.04. For
phylogenetic analysis, 75 gene sequences of 18S rRNA
from haptophytes were obtained from GenBank, including
a sequence of the Pavlovophyceae as an outgroup. The sequence of the specimen Tosa-3 was aligned together withthe sequences obtained from the GenBank by MAFFT(https://mafft.cbrc.jp/alignment/server/).
Phylogenetic trees were constructed based on Bayesian Inference (BI) using Mr. BAYES v3.2.7 (Huelsenbeck& Ronquist, 2001; Ronquist & Huelsenbeck, 2003) andon PhyML method using SeaView 5.04 (Guindon et al.,2010). Substitution models were selected using MrModeltest 2.2 (Nylander, 2004) and Modeltest 3.7 (Posada& Crandall, 1998) for BI and PhyML, respectively. For
the BI analysis, GTR + I + G model was selected by the
MrModeltest, and Markov chain Monte Carlo iterations
were carried out until 1 million generations when the average standard deviations of split frequencies were below
0.01, indicating convergence of the iterations. For the
PhyML analyses, a likelihood score (–lnL = 10496.6123)
was obtained under the GTR + I + G model with the following parameters: assumed nucleotide frequencies A =0.2318, C = 0.2215, G = 0.2919, T = 0.2548; substitution
rate A-C = 1.0902, A-G = 1.4920, A-T = 0.8190, C-G =0.7768, C-T = 4.3052, G-T = 1.0000; proportion of sites
assumed to be invariable = 0.5521; and rates for variable

Molecular phylogenetic affinity of Hayaster cf. perplexus based on 18S rDNA sequences

100

Plate 1

1

2

5 µm

3

5 µm

4

5 µm

5 µm

Plate 1: Light microscopic images of the isolate Tosa-3. 1–3. Plane-polarised light view. 4. Cross-polarised light view. Black and white arrows
indicate polygonal outline of distal shield and proximal shield, respectively. Dashed arrows show bright spots in central area of distal shield that may
correspond to the central process of Hayaster perplexus shown in Nishida (1979).

sites assumed to follow a gamma distribution with shapeparameter = 0.4539, were estimated by Modeltest 3.7(Posada & Crandall, 1998). Bootstrap analyses with 1000
replicates for PhyML analysis were applied to examine
the robustness and statistical reliability of the topologies(Felsenstein, 1985).
3. Results
3.1. Microscopic Study
The coccosphere of the isolate Tosa-3 was carefully observed under a polarised light microscope (Plate 1) before
the coccosphere was crushed for molecular study. It wasa partially disintegrated spherical coccosphere approxi-

mately 15 µm in diameter. The cell of the coccolithophore
was about 7 µm in diameter and only occupied a part ofthe internal coccosphere space; it had a prominent, dark
yellow chloroplast (Plate 1).
The coccosphere was monomorphic consisting of ≥30
large placoliths. The placoliths consisted of a broad (6–12µm), thin, concavo-convex distal shield and a much smaller proximal shield (white arrows in Plate 1, figures 3 and4), joined by a narrow tube. The distal shields were circular or subcircular in outline, but with a polygonal rim, andwere formed of 10–14 elements (black arrows in Plate 1,
figures 1–3). Very small (≤0.5 µm) bright spots were observed on the centre of the distal shields in both plane- and101

Hagino, Young, Morono
0.78/51 AJ544116 Calcidiscus leptoporus
KF422619 Calcidiscus leptoporus
0.88/38
0.77/43 AM491026 Oolithotus fragilis
0.96/51
AJ544115 Calcidiscus quadriperforatus
Calcidiscaceae
0.54/21
AJ544118 Umbilicosphaera sibogae
1.00/80
AJ544119 Umbilicosphaera foliosa
0.90/71
AM490993
Umbilicosphaera hulburtiana
1
AJ544117 Coccolithus braarudii
0.90/40
Coccolithaceae
AJ246261
Coccolithus pelagicus
0.73/79
FJ537310 Uncultured clone Biosope T33001

BI/PhyML
0.03

LC771592 Hayaster cf. perplexus isolate Tosa-3

AM490988 Calyptrosphaera sp.
AB183608 Calyptrosphaera sp.
AB183607 Helladosphaera sp.
AM490990 Calyptrosphaera sphaeroidea
1.00/84
AM490991 Helladosphaera sp.
AM490989 Holococcolithophorid sp.
0.91/46
AM490992
Calyptrosphaera
sp. LKM-2007-1
1.00/94
AJ544121 Pleurochrysis dentata
0.84/49
AJ246265 Pleurochrysis sp.
0.54/34
KF728655 Chrysotila stipitata
AJ544120 Pleurochrysis carterae
AJ246263 Pleurochrysis carterae
AM490973 Pleurochrysis pseudoroscoffensis
1.00/43
1.00/86
0.98/63
AM490981 Hymenomonas globosa
0.89/45
AM490982 Hymenomonas coronata
0.61/34
AM490980 Ochrosphaera verrucosa
FR865767 Ochrosphaera neapolitana
0.73/110.95/56
AM490979 Jomonlithus littoralis

Coccolithaceae

0.9/540.90/32

1.00/82

AM490985 Algirosphaera robusta
KF130396 Uncultured eukaryote clone ST8300 084
0.89/71
1.00/91 AM490987 Syracosphaera pulchra
KF422621 Syracosphaera pulchra
0.99/78
KF621001 Uncultured haptophyte clone 905 st12-14
0.59/12AM490986 Coronosphaera mediterranea
11.00/86
AM490998 Chrysotilala mellosa
AM490996 Isochrysis litoralis
AJ246266 Isochrysis galbana
1.00/99
AJ246276 Gephyrocapsa oceanica
KX056536 Gephyrocapsa parvula
FJ537341 Uncultured Chrysochromulina Biosope T60 034
KF130149 Uncultured eukaryote
LC595680 Braarudosphaera bigelowii
0.94/57
0.98/44
LC595681 Braarudosphaera bigelowii
AB636316 Tergestiella adriatica
AM490983 Helicosphaera carteri
1.00/80
AM490984 Scyphosphaera apsteinii
0.80/33
AF107084 Unidentified prymnesiophyte clone OLI26041
AF107088 Unidentified prymnesiophyte clone OLI51050
FN690514 Uncultured haptophyte
0.99/52
AB983345 Gladiolithus sp.

holococcolithbearing taxa

Coccolithales

AJ246262 Cruciplaccolithus neohelis
AB058348 Cruciplaccolithus neohelis

1.00/99

Pleurochrysidaceae

Hymenomonadaceae

0.6/16

AM491005 Prymnesium faveolatum
U40923 Prymnesium calathiferum
AM491001 Prymnesium zebrinum
1.00/32
AJ246269
Prymnesium parvum
10.96/68
1.00/84
AB183265 Hyalolithus neolepis
AJ246271
Prymnesium
kappa
0.56/86
AM491015 cf. Imantonia sp.
1.00/92
AM491014 Imantonia rotunda
0.98/32
AM491016 Pseudohaptolina arctica
0.94/77 AM491013 Haptolina fragaria
0.97/48
AM491012 Haptolina brevifila
1.00/88
AB058369 Haptolina ericina
AF107089 Unidentified prymnesiophyte clone OLI51059
AF107087 Unidentified prymnesiophyte clone OLI51033
0.87/32
AM491022 Chrysochromulina
spinifera
AJ246274 Chrysochromulina scutellum
1.00/71
1.00/99
AJ246273 Chrysochromulina campanulifera
AJ246279 Chrysochromulina trondsenii
AF107090 Unidentified prymnesiophyte clone OLI51076
AF107081 Unidentified prymnesiophyte clone OLI16010
AJ278035 Phaeocystis globosa
X77479 Phaeocystis antarctica

Syracosphaerales

Isochrysidales

Braarudosphaerales
Watznaueriales
Zygodiscales

Family Inc Sed

0.53/71

0.60/-

1.00/0.97/58

L34669 Pavlova salina

Prymnesiales

Phaeocystales

Pavlovales

Figure 1: Phylogenetic tree based on 18S rDNA sequences using the Bayesian Inference (BI) method. The number on each node indicates the posterior probability of BI analysis and bootstrap values from PhyML analysis. Solid circles indicate the clades supported by very high posterior probability(1.00) by BI analysis and bootstrap values (100%) by ML analysis.

cross-polarised light microscopy (black and white dottedarrows in Plate 1, figures 1, 3, and 4). The spots occasionally appeared brighter than the surrounding distal shieldelements under cross-polarised light (bottom two dottedarrows in Plate 1, figures 1–4), suggesting that the central area was filled with a calcified structure, and that theorientation of the calcite crystals forming the structuresdiffered from that of the distal shield.
3.1. Molecular phylogenetic study
The topography of the trees obtained by BI and PhyML
analyses resemble each other. Figure 1 shows only the BI

Molecular phylogenetic affinity of Hayaster cf. perplexus based on 18S rDNA sequences

tree with posterior probability obtained by BI analysis andbootstrap values from PhyML analysis. The sequence ofthe specimen Tosa-3 fell in the Coccolithales Clade withvery high posterior probability (1.0) and high bootstrap
support (94%). In the Coccolithales Clade, the sequence of
the isolate Tosa-3 made a subclade with the sequences ofCalcidiscaceae, Coccolithaceae, and holococcolith-bearing
taxa with very high posterior probability (1.0) and bootstrap support (99%). In the subclade, the isolate Tosa-3
clustered with Calcidiscaceae + Coccolithus pelagicus(Coccolithaceae) and an environmental sequence (Biosope
T33.001) with low posterior probability (0.73) and low
bootstrap support (79%).
4. Discussion
4.1. Identity of the coccolithophore
The observed coccosphere resembles Hayaster perplexus(Bramlette & Riedel, 1954) Bukry, 1973 in that it is amonomorphic coccosphere with large, thin, circular placoliths with small proximal shields, and in the polygonal outline of the distal shield. Typical H. perplexus coccoliths
have a solid tube and only a slight depression on either theproximal or distal surface (e.g. Young et al., 2003). However, an unusual H. perplexus specimen, which has a small
central process and pore on the polygonal distal shield, wasreported from the North Pacific Ocean (Nishida, 1979, pl.
4 fig. 2a; note that this image is also shown in Winter &Siesser, 1994, p. 120 and on the Nannotax3 website [Younget al., 2023]). The bright spots observed in the central areaof our isolate may correspond to the central processes observed in H. perplexus by Nishida (1979). Culture strainsof H. perplexus have never been established and there is no
information on the cause of the morphological variation in
the central structure of coccoliths of H. perplexus. Since
the distal shields of our isolate are polygonal, we are confident that our specimen belongs to Hayaster. However,
we have no evidence to determine whether the observed
central spots/processes reflect intraspecific morphologicalvariation of H. perplexus or pseudocryptic speciation. In
current taxonomy, the described species that most closely
resembles our specimen is H. perplexus; therefore, we refer
to our isolate Tosa-3 as Hayaster cf. perplexus.
4.2 Molecular phylogenetic placement of Hayastercf. perplexus
The families Coccolithaceae Poche, 1913 emend Young102& Bown, 1997 and Calcidiscaceae Young & Bown, 1997
are both characterised by diploid stages with monomorphic coccospheres of placolith coccoliths and haploidstages with monomorphic coccospheres of holococcoliths.
In both, the heterococcoliths have a distal shield formedof V-units (i.e. with sub-vertical crystallographic c-axes),
a proximal shield of R-units (i.e. with sub-radial crystallographic c-axes), and the locus of the proto-coccolithring is embedded within the coccolith (Young et al.,2004). Finally, both families have fossil records extending through the Cenozoic, from the Paleocene to the Recent. They were separated by Young & Bown (1997) on
the basis that in the Coccolithaceae, the V- and R-units areintertwined, forming an inner tube with an upper layer of
R-units and lower layer of V-units, whilst this configuration is absent in the Calcidiscaeae (see Figure 2). This
separation was consistent with a previous molecular phylogenetic study based on the tufA region with the Calcidiscaceae sequences forming a discrete clade with very
high posterior probabilities for MrBayes (MB = Bayesianinference) and low bootstrap support for Maximum Parsimony (MP) and Neighbor Joining (NJ) analyses (MB/MP/
NJ, 100/<50/62), albeit nesting within the Coccolithaceae(Medlin et al., 2008).
In this study, Calcidiscus, Oolithotus, and Umbilicosphaera made the Calcidiscaceae clade with very highCoccolithus

Calcidiscus
Figure 2: Schematic diagram of a cross section of placoliths of Coccolithus and Calcidiscus species. Colour indicates difference in orientationof elements: dark grey = V-unit and white = R-unit. Light grey zoneshows overlap of the V- and R-units. The open circles indicate the location of the proto-coccolith ring.

103

Hagino, Young, Morono

posterior probabilities for BI analyses and low bootstrap
support for PhyML analyses (BI/PhyML, 1.00/80%) similar to the tufA tree shown in Medlin et al. (2008). However, the sequence of Hayaster cf. perplexus unexpectedly
falls outside the Calcidiscaceae clade, together with thesequences of Coccolithaceae, although Hayaster perplexus has a Calcidiscaceae-type structure and has been included within the Calcidiscaceae in modern classifications(e.g. Young et al., 2003; Jordan et al., 2004). Our results
suggest that the Calcidiscaceae/Coccolithaceae division
may prove artificial, in which case the two families should
be recombined into a single family (Coccolithaceae), or
the definition of the Calcidiscaceae should be emended to
exclude Hayaster. Our data are not sufficiently robust to
support a change of classification at this stage, but they dosuggest that further investigation using additional taxa or
additional genetic systems would be worthwhile.

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Acknowledgements
This research was supported by Grant-in-aid for Scientific Research from the Japan Society for the Promotionof Sciences (No. 20H04325). We acknowledge the staffof the Usa Marine Biological Institute, Kochi University
for their assistance with sampling. We would like to thankDr. El Mahdi Bendif and an anonymous reviewer for theirconstructive reviews.

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